EP0687180B1 - Procedes de detection et de traitement de personnes presentant des cellules anormales exprimant les antigenes peptidiques hla-a2/tyrosinase - Google Patents

Procedes de detection et de traitement de personnes presentant des cellules anormales exprimant les antigenes peptidiques hla-a2/tyrosinase Download PDF

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EP0687180B1
EP0687180B1 EP94905393A EP94905393A EP0687180B1 EP 0687180 B1 EP0687180 B1 EP 0687180B1 EP 94905393 A EP94905393 A EP 94905393A EP 94905393 A EP94905393 A EP 94905393A EP 0687180 B1 EP0687180 B1 EP 0687180B1
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cells
leu
composition
hla
molecule
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EP0687180A4 (fr
EP0687180A1 (fr
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Thierry Boon-Falleur
Vincent Brichard
Aline Van Pel
Etienne De Plaen
Pierre Coulie
Jean-Christophe Renauld
Thomas Wolfel
Bernard Lethe
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Ludwig Institute for Cancer Research New York
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Ludwig Institute for Cancer Research Ltd
Ludwig Institute for Cancer Research New York
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/18—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with another compound as one donor, and incorporation of one atom of oxygen (1.14.18)
    • C12Y114/18001—Tyrosinase (1.14.18.1)
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
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    • Y10S436/813—Cancer
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y10S530/827—Proteins from mammals or birds
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Definitions

  • This invention relates to various therapeutic methodologies derived from the recognition that certain abnormal cells present complexes of human leukocyte antigens and peptides derived from tyrosinase on their surfaces. In addition, it relates to the ability to identify those individuals diagnosed with conditions characterized by cellular abnormalities whose abnormal cells present this complex, the presented peptides, and the ramifications thereof.
  • the process by which the mammalian immune system recognizes and reacts to foreign or alien materials is a complex one.
  • An important facet of the system is the T cell response. This response requires that T cells recognize and interact with complexes of cell surface molecules, referred to as human leukocyte antigens ("HLA”), or major histocompatibility complexes ("MHCs”), and peptides.
  • HLA human leukocyte antigens
  • MHCs major histocompatibility complexes
  • peptides are derived from larger molecules which are processed by the cells which also present the HLA/MHC molecule. See in this regard Male et al., Advanced Immunology (J.P. Lipincott Company, 1987), especially chapters 6-10.
  • T cell and complexes of HLA/peptide are restricted, requiring a T cell specific for a particular combination of an HLA molecule and a peptide. If a specific T cell is not present, there is no T cell response even if its partner complex is present. Similarly, there is no response if the specific complex is absent, but the T cell is present.
  • This mechanism is involved in the immune system's response to foreign materials, in autoimmune pathologies, and in responses to cellular abnormalities. Recently, much work has focused on the mechanisms by which proteins are processed into the HLA binding peptides. See, in this regard, Barinaga, Science 257: 880 (1992); Fremont et al.. Science 257: 919 (1992); Matsumura et al., Science 257: 927 (1992); Latron et al., Science 257: 964 (1992).
  • T cells recognize cellular abnormalities has also been implicated in cancer.
  • PCT Publication No. WO 92/20356 filed May 22, 1992, published on November 26, 1992.
  • a family of genes is disclosed, which are processed into peptides which, in turn, are expressed on cell surfaces, which can lead to lysis of the tumor cells by specific CTLs.
  • the genes are said to code for "tumor rejection antigen precursors or "TRAP” molecules, and the peptides derived therefrom are referred to as "tumor rejection antigens" or "TRAs”. See Traversari et al., Immunogenetics 35: 145 (1992); van der Bruggen et al., Science 254: 1643 (1991), for further information on this family of genes.
  • nonapeptides which bind to the HLA-A1 molecule.
  • the reference teaches that given the known specificity of particular peptides for particular HLA molecules, one should expect a particular peptide to bind one HLA molecule, but not others. This is important, because different individuals possess different HLA phenotypes. As a result, while identification of a particular peptide as being a partner for a specific HLA molecule has diagnostic and therapeutic ramifications, these are only relevant for individuals with that particular HLA phenotype. There is a need for further work in the area, because cellular abnormalities are not restricted to one particular HLA phenotype, and targeted therapy requires some knowledge of the phenotype of the abnormal cells at issue.
  • the enzyme tyrosinase catalyzes the reaction converting tyrosine to dehydroxyphenylalanine or "DOPA" and appears to be expressed selectively in melanocytes (Muller et al., EMBOJ 7: 2715 (1988)).
  • DOPA dehydroxyphenylalanine
  • An early report of cDNA for the human enzyme is found in Kwon, U.S. Patent No. 4,898,814.
  • tyrosinase may be treated in a manner similar to a foreign antigen or a TRAP molecule - i.e., it has now been found that in certain cellular abnormalities, such as melanoma, tyrosinase is processed and a peptide derived therefrom forms a complex with HLA molecules on certain abnormal cells. These complexes are recognized by cytolytic T cells ("CTLs”), which then lyse the presenting cells.
  • CTLs cytolytic T cells
  • the present invention provides an isolated peptide consisting of the amino acid sequence Met Leu Leu Ala Val Leu Tyr Cys Leu Leu.
  • the present invention provides a method for determining presence of melanoma cell specific cytolycic T cells in a sample as a diagnosis of melanoma in a subject from whom said sample has been taken, comprising contacting a mononuclear blood cell containing sample from a subject of interest with melanoma cells which present, on their surface, complexes of HLA-A2 molecules and the peptide Met Leu Leu Ala Val Leu Tyr Cys Leu Leu, and determining one of (i) proliferation of cytolytic T cells in said mononuclear blood cell containing sample and (ii) lysis of said melanoma cells, as a determination of presence of melanoma cell specific cytolytic T cells in said sample, said presence being indicative of melanoma in said subject.
  • the present invention provides a composition for use in a therapeutic treatment comprising an isolated peptide in accordance with the first aspect of the invention.
  • the invention provides a composition for use in a therapeutic treatment, comprising a complex of an HLA molecule and a peptide having the amino acid sequence Met Leu Leu Ala Val Leu Tyr Cys Leu Leu.
  • the invention provides a composition
  • a composition comprising cells presenting a complex of an HLA molecule and a peptide having the amino acid sequence Met Leu Leu Ala Val Leu Tyr Cys Leu Leu on their surface, wherein said cells have been transfected with one or both of (i) a nucleic acid molecule which codes for an HLA molecule and (ii) a nucleic acid molecule which codes for tyrosinase, or a portion thereof that includes the amino acid sequence Met Leu Leu Ala Val Leu Tyr Cys Leu Leu.
  • the present invention provides a method of generating Cytolytic T cells specific for a complex comprising an HLA molecule and a peptide having the amino acid sequence Met Leu Leu Ala Val Leu Tyr Cys Leu Leu, said method comprising the step of contacting a sample of blood cells to cells presenting said complex.
  • SK 29-MEL also referred to in the literature as SK MEL-29
  • LB24-MEL which have been available to researchers for many years, were used in the following experiments.
  • Samples containing mononuclear blood cells were taken from patients AV and LB24-MEL (these patients were also the source of SK 29-MEL and LB24-MEL, respectively).
  • the melanoma cell lines were contacted to the mononuclear blood cell containing samples.
  • the mixtures were observed for lysis of the melanoma cell lines, this lysis indicating that cytolytic T cells ("CTLs") specific for a complex of peptide and HLA molecule presented by the melanoma cells were present in the sample.
  • CTLs cytolytic T cells
  • the lysis assay employed was a chromium release assay following Herin et al., Int. J. Cancer 39:390-396 (1987), the disclosure of which is incorporated by reference. The assay, however, is described herein.
  • the target melanoma cells were grown in vitro , and then resuspended at 10 7 cells/ml in DMEM, supplemented with 10 mM HEPES and 30% FCS, and incubated for 45 minutes at 37°C with 200 ⁇ Ci/ml of Na( 51 Cr)O 4 . Labelled cells were washed three times with DMEM, supplemented with 10 mM Hepes.
  • % 51 Cr release (ER-SR) (MR-SR) x 100 where ER is observed, experimental 51 Cr release, SR is spontaneous release measured by incubating 10 3 labeled cells in 200 ul of medium alone, and MR is maximum release, obtained by adding 100 ul 0.3% Triton X-100 to target cells.
  • Figure 1 presents the results of these assays, in panels A, B, G and I. Specifically, it will be seen that both CTLs lysed both melanoma cell lines, and that there was no lysis of the K562 and EBV-B cell lines.
  • the tested lines are known to be of type HLA-A2, and the results suggested that the CTLs are specific for a complex of peptide and HLA-A2. This suggestion was verified by testing a variant of SK 29-MEL which has lost HLA-A2 expression. Figure 1, panel F shows these results. Neither clone lysed the HLA-loss variant. When the variant was transfected with the HLA-A2 gene of SK29-MEL, however, and retested, lysis was observed. Thus, it can be concluded that the presenting molecule is HLA-A2.
  • TRIP tumor rejection antigen precursor
  • the RNA was isolated using an oligo-dT binding kit, following well recognized techniques. Once the total RNA was secured, it was transcribed into cDNA, again using standard methodologies.
  • the cDNA was then ligated to EcoRI adaptors and cloned into the EcoRI site of plasmid pcDNA-I/Amp, in accordance with manufacturer's instructions.
  • the recombinant plasmids were then electroporated into JM101.
  • E . coli electroporated into JM101.
  • E . coli electroporation conditions: 1 pulse at 25 ⁇ farads, 2500 V).
  • the transfected bacteria were selected with ampicillin (50 ⁇ g/ml), and then divided into 700 pools of 200 clones each. Each pool represented about 100 different cDNAs, as analysis showed that about 50% of plasmids contained an insert. Each pool was amplified to saturation, and plasmid DNA was isolated via alkaline lysis, potassium acetate precipitation and phenol extraction, following Maniatis et al., in Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y., 1982). Cesium gradient centrifugation was not used.
  • the amplified plasmids were then transfected into eukaryotic cells.
  • Samples of COS-7 cells were seeded, at 15,000 cells/well into tissue culture flat bottom microwells, in Dulbeco's modified Eagles Medium ("DMEM") supplemented with 10% fetal calf serum. The cells were incubated overnight at 37°C, medium was removed and then replaced by 30 ⁇ l/well of DMEM medium containing 10% Nu serum, 400 ⁇ g/ml DEAE-dextran, 100 ⁇ M chloroquine, 100 ng of plasmid pcDNA-I/Amp-A2 and 100 ng of DNA of a pool of the cDNA library described supra.
  • DMEM Dulbeco's modified Eagles Medium
  • Plasmid pcDNA-I/Amp-A2 contains the HLA-A2 gene from SK29-MEL. Following four hours of incubation at 37°C, the medium was removed, and replaced by 50 ⁇ l of PBS containing 10% DMSO. This medium was removed after two minutes and replaced by 200 ⁇ l of DMEM supplemented with 10% of FCS.
  • COS cells were incubated for 48 hours at 37°C. Medium was then discarded, and 2000 cells of either of the described CTL clones were added, in 100 ⁇ l of Iscove medium containing 10% pooled human serum. When clone 210/9 was used, the medium was supplemented with 25 U/ml of IL-2. Supernatant was removed after 24 hours, and TNF content was determined in an assay on WEHI cells, as described by Traversari et al., Immunogenetics 35: 145-152 (1992), the disclosure of which is incorporated by reference.
  • WEHI OD's correspond to 24 pg/ml of TNF for cDNA and HLA-A2, versus 2.3 pg/ml for the control.
  • a normally occurring molecule i.e., tyrosinase
  • tyrosinase may act as a tumor rejection antigen precursor and be processed to form a peptide tumor rejection antigen which is presented on the surface of a cell, in combination with HLA-A2, thereby stimulating lysis by CTL clones.
  • the nucleic sequence of the identified molecule is presented as SEQ ID NO: 1.
  • Hunt discusses nonapeptides, where either Leu or Ile is always found at the second position, Leu being the "dominant residue".
  • the ninth residue is described as always being a residue with an aliphatic hydrocarbon side chain. Val is the dominant residue at this position.
  • Hunt discusses a strong signal for Leu and an intermediate signal for Met at the second position, one of Val, Leu, Ile or Thr at position 6, and Val or Leu at position 9, with Vat being particularly strong.
  • nonapeptides were synthesized and then tested to see if they could sensitize HLA-A2 presenting cells. To do so, tyrosinase loss variant cell lines SK29-MEL 1.218 and T202LB were used.
  • Varying concentrations of the tested peptides were added to the cell lines, together with either of cytolytic T cell clone CTL IVSB or cytolytic T cell clone CTL 2/9.
  • Prior work, described supra, had established that the former clone lysed tyrosinase expressing cells which present HLA-A2, and that the latter did not.
  • the tyrosinase loss variants were incubated for one hour in a solution containing 51 Cr, at 37°C, either with or without anti HLA-A2 antibody MA2.1, which was used to stabilize empty HLA-A2 molecules.
  • cells were washed four times, and then incubated with varying dilutions of the peptides, from 100 ⁇ M down to 0.01 ⁇ M. After 30 minutes, effector cells were added at an E/T ratio of 40/1 and four hours later, 100 ⁇ of supernatant were collected and radioactivity counted.
  • Figure 4 shows the results obtained with nonapeptide Tyr Met Asn Gly Thr Met Ser Gln Val. (SEQ ID NO: 2).
  • SEQ ID NO: 2 corresponds to residues 1129-1155 of the cDNA sequence for tyrosinase presented in SEQ ID NO: 1.
  • Complexes of HLA-A2 and this peptide are recognized by CTL clone CTL IVSB.
  • CTL clone CTL 210/9 did not recognize the complexes of HLA-A2 and the peptide of SEQ ID NO: 2, although it did recognize complexes of HLA-A2 and a tyrosinase derived peptide.
  • tyrosinase is processed to at least one additional peptide which, when presented by HLA-A2 molecules, is recognized by CTL clones.
  • a second fragment, corresponding to bases 451-1158 of SEQ ID NO: 1 was transfected into COS cells together with a gene for HLA-A2, and TNF release assays were carried out.
  • This sequence provoked TNF release from clone SK29-CTL IVSB (20 pg/ml), but not from LB24-CTL 210/9 (3.8 pg/ml).
  • the tyrosinase derived peptide coded for by cDNA fragment 1-451 was analyzed for consensus sequences known to bind HLA-A2.
  • the peptides corresponding to these consensus sequences were synthesized, and tested for their ability to sensitize HLA-A2 presenting cells. To do so, two tyrosinase negative melanoma cell lines were used (i.e., NA8-MEL, and MZ2-MEL 2.2 transfected with HLA-A2), and cell line T2, as described by Salter et al, Immunogenetics 21: 235-246 (1985), were used.
  • the cells were incubated with 51 Cr, and monoclonal antibody MA.2.1, which is specific to HLA-A2 for 50 minutes at 37°C, followed by washing (see Bodmer et al., Nature 342: 443-446 (1989), the disclosure of which is incorporated by reference in its entirety).
  • Target cells were incubated with various concentrations of the peptides, and with either of LB 24-CTL clones 210/5 or 210/9. The percent of chromium release was measured after four hours of incubation.
  • the peptide Met Leu Leu Ala Val Leu Tyr Cys Leu Leu (SEQ ID NO: 3) was found to be active.
  • tyrosinase gene encodes an antigen recognized by autologous CTLs on most HLA-A2 melanoma. Expression of this gene on sublines of cell line MZ2-MEL was tested by PCR amplification. Clone MZ2-MEL.43 was found to be positive, whereas other MZ2-MEL clones, such as MZ2-MEL.3.0 were negative. Correlation of expression of the tyrosinase gene, and antigen MZ2-C, suggested that MZ2-C might be a tumor rejection antigen derived from tyrosinase, and presented by an HLA molecule expressed by MZ2-MEL. This cell line does not express HLA-A2, which would indicate that if a tyrosinase derived peptide were presented as a TRA, a second HLA molecule was implicated.
  • HLA-A29, HLA-B37, HLA-B 44.02, and HLA-C clone 10 were isolated from an MZ2-MEL.43 cDNA library, and then cloned into expression vector pcDNAI/Amp. Recipient COS 7 cells were then transfected with one of these constructs or a construct containing HLA-A1, plus cDNA coding for tyrosinase (SEQ ID NO: 1). The contransfection followed the method set forth above.
  • a sample of cells such as blood cells
  • a cell presenting the complex and capable of provoking a specific CTL to proliferate are contacted to a cell presenting the complex and capable of provoking a specific CTL to proliferate.
  • the target cell can be a transfectant, such as a COS cell of the type described supra. These transfectants present the desired complex on their surface and, when combined with a CTL of interest, stimulate its proliferation.
  • vectors containing the genes of interest i.e., pcDNA-1/Amp1 (HLA-A2), and p123.B2 (human tyrosinase), have been deposited in accordance with the Budapest Treaty at the Institut Pasteur, under Accession Numbers I1275 and I1276, respectively.
  • COS cells such as those used herein are widely available, as are other suitable host cells.
  • the foregoing therapy assumes that at least some of the subject's abnormal cells present one or more of the HLA/tyrosinase derived peptide complexes. This can be determined very easily. For example CTLs are identified using the transfectants discussed supra , and once isolated, can be used with a sample of a subject's abnormal cells to determine lysis in vitro . If lysis is observed, then the use of specific CTLs in such a therapy may alleviate the condition associated with the abnormal cells.
  • a less involved methodology examines the abnormal cells for HLA phenotyping, using standard assays, and determines expression of tyrosinase via amplification using, e.g., PCR. The fact that a plurality of different HLA molecules present TRAs derived from tyrosinase increases the number of individuals who are suitable subjects for the therapies discussed herein.
  • Adoptive transfer is not the only form of therapy that is available in accordance with the invention.
  • CTLs can also be provoked in vivo , using a number of approaches.
  • One approach i.e., the use of non-proliferative cells expressing the complex, has been elaborated upon supra.
  • the cells used in this approach may be those that normally express the complex, such as irradiated melanoma cells or cells transfected with one or both of the genes necessary for presentation of the complex. Chen et al., Proc. Natl. Acad. Sci. USA 88: 110-114 (January, 1991) exemplifies this approach, showing the use of transfected cells expressing HPVE7 peptides in a therapeutic regime.
  • Various cell types may be used.
  • vectors carrying one or both of the genes of interest may be used.
  • Viral or bacterial vectors are especially preferred.
  • the gene of interest is carried by, e.g., a Vaccinia virus or the bacteria BCG, and the materials de facto "infect" host cells.
  • the cells which result present the complex of interest, and are recognized by autologous CTLs, which then proliferate.
  • a similar effect can be achieved by combining tyrosinase itself with an adjuvant to facilitate incorporation into HLA-A2 presenting cells.
  • the enzyme is then processed to yield the peptide partner of the HLA molecule.
  • abnormal cells and "cellular abnormalities”. These terms are employed in their broadest interpretation, and refer to any situation where the cells in question exhibit at least one property which indicates that they differ from normal cells of their specific type. Examples of abnormal properties include morphological and biochemical changes, e.g. Cellular abnormalities include tumors, such as melanoma, autoimmune disorders, and so forth.
  • the invention also provides a method for identifying precursors to CTL targets. These precursors are referred to as tumor rejection antigens when the target cells are tumors, but it must be pointed out that when the cell characterized by abnormality is not a tumor, it would be somewhat misleading to refer to the molecule as a tumor rejection antigen.
  • the method involves identifying a cell which is the target of a cytolytic T cell of the type discussed supra . Once such a cell is identified, total RNA is converted to a cDNA library, which is then transfected into a cell sample capable of presenting an antigen which forms a complex with a relevant HLA molecule.
  • transfectants are contacted with the CTL discussed supra , and again, targeting by the CTL is observed (lysis and/or TNF production). These transfectants which are lysed are then treated to have the cDNA removed and sequenced, and in this manner a precursor for an abnormal condition, such as a tumor rejection antigen precursor, can be identified.
  • an abnormal condition such as a tumor rejection antigen precursor

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Claims (35)

  1. Un peptide isolé consistant dans la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu.
  2. Une méthode pour la détermination de la présente de cellules cytolytiques T spécifiques aux cellules de mélanomes dans un échantillon, en vue du diagnostic du mélanome dans un sujet sur lequel ledit échantillon a été prélevé, méthode consistant à mettre en contact un échantillon contenant des cellules sanguines mononucléaires provenant d'un sujet en cause avec des cellules de mélanome qui présentent sur leur surface des complexes de molécules HLA-A2 et du peptide Met Leu Leu Ala Val Leu Tyr Cys Leu Leu et à déterminer (i) soit la prolifération de cellules cytolytiques T dans ledit échantillon contenant des cellules sanguines mononucléaires, (ii) soit la lyse desdites cellules de mélanome, en tant que détermination de la présence de cellules cytolytiques T spécifiques aux cellules de mélanome dans ledit échantillon, ladite présence indiquant un mélanome dans ledit sujet.
  3. Une méthode selon la revendication 2 consistant à déterminer la prolifération de cellules cytolytiques T en mesurant le dégagement du facteur de nécrose tumorale.
  4. Une méthode selon les revendications 2 ou 3, consistant à déterminer la lyse desdites cellules en déterminant le dégagement de chrome radiomarqué.
  5. Une méthode selon l'une quelconque des revendications 2-4, dans laquelle lesdites cellules qui présentent lesdits complexes ont été transfectées par au moins l'une de (i) une molécule d'acide nucléique qui code pour une molécule HLA et (ii) une molécule d'acide nucléique qui code pour la tyrosinase.
  6. Une méthode selon l'une quelconque des revendications 2-4, dans laquelle lesdites cellules ont été transfectées à la fois par (i) une molécule d'acide nucléique qui code pour une molécule HLA et (ii) une molécule d'acide nucléique qui code pour la tyrosinase.
  7. Une composition destinée à être utilisée dans un traitement thérapeutique, comprenant un peptide isolé selon la revendication 1.
  8. Une composition destinée à être utilisée dans un traitement thérapeutique, comprenant un complexe d'une molécule HLA et d'un peptide ayant la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu.
  9. Une composition comprenant des cellules présentant un complexe d'une molécule HLA et d'un peptide ayant la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu sur leur surface, dans laquelle lesdites cellules ont été transfectées avec l'un ou les deux de (i) une molécule d'acide nucléique qui code pour une molécule HLA et (ii) une molécule d'acide nucléique qui code pour la tyrosinase, ou une portion de celle-ci qui inclut la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu.
  10. Une composition selon la revendication 8, comprenant des cellules présentant ledit complexe sur leur surface.
  11. Une composition selon la revendication 10, dans laquelle les cellules sont des transfectants.
  12. Une composition selon la revendication 11, dans laquelle les cellules ont été transfectées par l'une ou les deux de (i) une molécule d'acide nucléique qui code pour une molécule HLA et (ii) une molécule d'acide nucléique qui code pour la tyrosinase, ou une portion de celle-ci qui inclut la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu.
  13. Une composition selon la revendication 9 ou la revendication 12, dans laquelle lesdites cellules ont été transfectées avec l'un ou les deux des vecteurs pcDNA-1/Ampl et p123.B2 déposés à l'institut Pasteur sous les numéros d'accès I1275 et I1276 respectivement.
  14. Une composition selon l'une quelconque des revendications 9 à 13, dans laquelle les cellules sont non proliférantes.
  15. Une composition selon l'une quelconque des revendications 9 à 14, dans laquelle les cellules sont des cellules de mélanome.
  16. Une composition destinée à être utilisée dans un traitement thérapeutique, comprenant un vecteur capable de provoquer une réponse de cellules cytolytiques T à des cellules présentant un complexe d'une molécule HLA et d'un peptide ayant la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu, ledit vecteur transportant (i) une molécule d'acide nucléique qui code pour la tyrosinase ou une portion de celle-ci qui inclut la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu et, facultativement (ii) une molécule d'acide nucléique qui code pour la molécule HLA.
  17. Une composition selon l'une quelconque des revendications 7 à 15, dans laquelle ladite composition est destinée à être utilisée dans la thérapie d'une tumeur.
  18. Une composition selon la revendication 12, dans laquelle ladite tumeur est un mélanome.
  19. Une composition selon l'une quelconque des revendications 7, 8, 14 et 16, dans laquelle ladite composition est destinée à être utilisée dans un traitement thérapeutique, impliquant l'administration de la composition à un sujet présentant une anomalie cellulaire caractérisée par le fait que certaines des cellules anormales présentent un complexe d'une molécule HLA comprenant un peptide ayant la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu.
  20. Une composition selon l'une quelconque des revendications 9 à 15, dans laquelle ladite composition est destinée à être utilisée dans un traitement thérapeutique faisant intervenir la mise en contact d'un échantillon de cellules sanguines avec la composition en vue de générer des cellules cytolytiques T spécifiques pour un complexe d'une molécule HLA avec un peptide ayant la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu et l'administration des cellules cytolytiques T à un sujet présentant une anomalie cellulaire caractérisée par le fait que certaines des cellules anormales présentant le complexe.
  21. Une composition selon l'une quelconque des revendications 8 à 20, dans laquelle la molécule HLA est une molécule HLA-A2.
  22. Utilisation d'une composition telle que définie dans l'une quelconque des revendications 7 à 21 dans la préparation d'un médicament destiné à être utilisé dans la thérapie d'une tumeur.
  23. Utilisation d'une composition telle que définie dans l'une quelconque des revendications 7 à 21 dans la préparation d'un médicament destiné à être utilisé dans la thérapie d'une anomalie cellulaire qui est caractérisée par le fait que certaines des cellules anormales présentent un complexe d'une molécule HLA et d'un peptide ayant la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu.
  24. Une utilisation selon la revendication 22, dans laquelle la tumeur est un mélanome.
  25. Une utilisation selon l'une quelconque des revendications 22 à 24, dans laquelle la thérapie implique l'administration de cellules cytolytiques T spécifiques vis-à-vis d'un complexe d'une molécule HLA et d'un peptide ayant la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu à un sujet.
  26. Une utilisation selon la revendication 25 dans laquelle les cellules cytolytiques T sont générées in vitro.
  27. Une utilisation selon la revendication 26 dans laquelle la composition est telle que définie dans l'une quelconque des revendications 9 à 15 et les cellules cytolytiques T sont générées par mise en contact d'un échantillon de cellules sanguines avec la composition.
  28. Une utilisation selon l'une quelconque des revendications 22 à 24, dans laquelle la thérapie implique une réponse CTL générée in vivo.
  29. Une utilisation selon la revendication 28, dans laquelle ledit traitement implique l'administration de la composition à un sujet présentant une anomalie cellulaire caractérisée par te fait que certaines des cellules anormales présentent un complexe d'une molécule HLA et d'un peptide ayant la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu.
  30. Une utilisation selon l'une quelconque des revendications 23 à 29, dans laquelle la molécule HLA est une molécule HLA-A2.
  31. Une méthode pour générer des cellules cytolytiques T spécifiques pour un complexe comprenant une molécule HLA et un peptide ayant la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu, ladite méthode comprenant l'étape consistant à mettre en contact un échantillon de cellules sanguines avec des cellules présentant ledit complexe.
  32. Une méthode selon la revendication 31, dans laquelle la molécule HLA est HLA-A2.
  33. Une méthode selon la revendication 31 ou la revendication 32 dans laquelle les cellules ont été transfectées avec au moins l'une de (i) une molécule d'acide nucléique qui code pour HLA-A2 et (ii) une molécule d'acide nucléique qui code pour la tyrosinase, ou une portion de celle-ci qui inclut la séquence d'aminoacides Met Leu Leu Ala Val Leu Tyr Cys Leu Leu.
  34. Une méthode de préparation d'une composition destinée à être utilisée dans un traitement thérapeutique, comprenant la mise en oeuvre d'une méthode selon l'une quelconque des revendications 31 à 33.
  35. Une méthode selon la revendication 34, dans laquelle ledit traitement est celui du cancer, de préférence le mélanome.
EP94905393A 1992-12-22 1993-12-14 Procedes de detection et de traitement de personnes presentant des cellules anormales exprimant les antigenes peptidiques hla-a2/tyrosinase Expired - Lifetime EP0687180B1 (fr)

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US5471493A 1993-04-28 1993-04-28
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US81673 1993-06-23
US08/081,673 US5487974A (en) 1992-12-22 1993-06-23 Method for detecting complexes containing human leukocyte antigen A2 (HLA-A2) molecules and a tyrosinase drived peptide on abnormal cells
PCT/US1993/012200 WO1994014459A1 (fr) 1992-12-22 1993-12-14 Procedes de detection et de traitement de personnes presentant des cellules anormales exprimant les antigenes peptidiques hla-a2/tyrosinase

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US5519117A (en) * 1992-12-22 1996-05-21 Ludwig Institute For Cancer Research Isolated, tyrosinase derived peptides and uses thereof
US5843648A (en) * 1995-01-10 1998-12-01 The United States Of America As Represented By The Secretary, Department Of Health And Human Services P15 and tyrosinase melanoma antigens and their use in diagnostic and therapeutic methods
US5821122A (en) * 1995-06-07 1998-10-13 Inserm (Institute Nat'l De La Sante Et De La Recherche . .) Isolated nucleic acid molecules, peptides which form complexes with MHC molecule HLA-A2 and uses thereof
UA68327C2 (en) 1995-07-04 2004-08-16 Gsf Forschungszentrum Fur Unwe A recombinant mva virus, an isolated eukaryotic cell, infected with recombinant mva virus, a method for production in vitro of polypeptides with use of said cell, a method for production in vitro of virus parts (variants), vaccine containing the recombinant mva virus, a method for immunization of animals
US6951917B1 (en) 1995-09-26 2005-10-04 The United States Of America As Represented By The Department Of Health And Human Services MHC-class II restricted melanoma antigens and their use in therapeutic methods
US7501501B2 (en) 1995-09-26 2009-03-10 The United States Of America As Represented By The Secretary Department Of Health And Human Services MHC-Class II restricted melanoma antigens and their use in therapeutic methods
US7019112B1 (en) 1996-03-19 2006-03-28 University Of Virginia Patent Foundation Peptides recognized by melanoma-specific A1-, A2- and A3-restricted cytoxic lymphocytes, and uses therefor
AU712820B2 (en) * 1996-03-19 1999-11-18 University Of Virginia Patent Foundation Peptides recognized by melanoma-specific A1-, A2- and A3-restricted cytotoxic lymphocytes, and uses therefor
EP0910646A1 (fr) * 1996-06-25 1999-04-28 The Ludwig Institute for Cancer Research Antigene anticancereux sous forme de phosphorylase agissant sur le glycogene cerebral
WO1998033810A2 (fr) * 1997-01-30 1998-08-06 University Of Virginia Patent Foundation Peptides appauvris en cysteine reconnus par des lymphocytes cytotoxiques limites par a3 et leurs mises en application
US20030138808A1 (en) * 1998-02-19 2003-07-24 Simard John J.L. Expression vectors encoding epitopes of target-associated antigens
US20030215425A1 (en) * 2001-12-07 2003-11-20 Simard John J. L. Epitope synchronization in antigen presenting cells
US7470428B2 (en) 2002-01-30 2008-12-30 The Brigham And Women's Hospital, Inc. Compositions and methods related to TIM-3, a Th1-specific cell surface molecule
CN101057145B (zh) 2004-06-23 2014-10-22 得克萨斯系统大学评议会 使用双粒子复合体检测生物分子的方法和组合物
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US4898814A (en) * 1986-10-06 1990-02-06 Donald Guthrie Foundation For Medical Research, Inc. A cDNA clone for human tyrosinase
EP0507772A1 (fr) * 1989-04-26 1992-10-14 Sloan-Kettering Institute For Cancer Research Cellule eucaryote non melanocytique exprimant de maniere constitutive la tyrosinase humaine biologiquement active, et son utilisation
US5342774A (en) 1991-05-23 1994-08-30 Ludwig Institute For Cancer Research Nucleotide sequence encoding the tumor rejection antigen precursor, MAGE-1
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